Using the MLL translocation as an MRD marker in infant ALL

1. Using the MLL translocation as an MRD marker in infant ALL Rich Hathway Bristol Genetics Laboratories

2. Acute Lymphoblastic Leukaemia (ALL) • ALL is a form of cancer that affects the lymphocyte producing cells in the bone marrow • In ALL there is an accumulation in the bone marrow of immature lymphocyte precursor cells called blasts • ALL is the most common cause of cancer in childhood affecting ~400 children per year in the UK Diagnosis: • 1012 leukaemic cells present at the time of diagnosis • i.e. 1,000,000,000,000 (million million cells) • Easily detected under a light microscope Remission: • Remission is defined as leukaemic cells being no longer detectable by light microscopy. • At this stage 5% of cells may actually be leukaemic, meaning that we are failing to detect 5 x 1010 leukaemic cells in the body i.e. 50,000,000,000 cells.

3. Minimal Residual Disease (MRD) • Level of disease still present that can’t be seen using a light microscope • Three methods currently available: • Flow cytometric immunophenotyping • Utilises tumour associated aberrant immunophenotypes e.g. Presence of myeloid markers on leukaemia blasts • Reverse transcriptase (RT) PCR • Utilises tumour specific RNA targets e.g. Fusion gene transcripts • Real-time Quantitative (RQ) PCR • Utilises patient-specific gene rearrangements e.g. Immunoglobulin and T-cell receptor gene rearrangements • Important considerations: applicability, stability, sensitivity & quantitation • RQ-PCR methodology is method of choice in most European MRD-based clinical trials • Analysis of minimal residual disease during treatment has been shown to be a very good indicator of outcome.

4. Minimal Residual Disease (MRD) 1012 Relapse 1011 Haematologic remission Detection limit of cytomorphology MRD Relative frequency of leukaemic cells 107 Detection limit of PCR technique “cure” 0 Follow-up In years MRD Analysis

5. Infant ALL • Infant ALL is very rare comprising ~4% of all childhood ALL cases • Approximately 15 cases per year in the UK • Age at diagnosis <365 days • Children with ALL >1yr old have excellent outcomes • Eligible for the ALL2003 clinical trial • Event free survival (EFS) >80% • Infant ALL have very poor prognosis • EFS ~40% (mid 1990s) • EFS varies with risk group

6. Interfant 06 clinical trial • International trial with 20 countries participating and a buddy system covering majority of the world • Phase III design where patients are stratified into three risk groups based on MLL gene status, age and white blood cell (WBC) count. • Low risk (LR): MLL germline; (~20% patients) • 2 year EFS 87% • High risk (HR): MLL rearranged & <6 months old & WBC ≥300 x 109/L (~21% patients) • 2 year EFS 15.9% • Medium risk (MR): all other cases, (59% patients) • 2 year EFS 49.5%

7. Interfant 06 treatment schematic Primary aim of Interfant-06 is to establish whether an additional block of AML induction therapy is beneficial MRD analysis ~ week 16

8. Mixed lineage leukaemia gene (MLL) is located on chromosome 11q23 ( ) MLL gene is involved in ~10% of chromosomal translocations in acute leukaemias In infant ALL MLL translocations are involved in >50% cases. Most MLL fusion genes have a poor clinical prognosis associated with them. The MLL fusion gene is potentially a strong MRD marker. At least 85 MLL fusion genes have been identified (e.g. & ) Reciprocal translocations Partial tandem duplications Deletions Inversions Insertions 3 way translocations Rare but seen once in our lab [46,XX, t(9;11;12) (p22;q23;q24)] MLL Chromosomal Translocations C.Meyer et al. Leukemia 2006

9. Sequencing of the MLL rearrangement is achieved by long distance inverse PCR (LDI-PCR) Mutant DNA is restricted and circularised Non rearranged MLL genes form 2 equal rings comprising the breakpoint cluster region (bcr) (B) Rearranged MLL genes form three different rings (C) A circularised MLL bcr A circularised der(11) A circularised der (Translocation partner) Using MLL gene specific oligonucleotides (A-L) PCR is performed on the circularised bcr and derivatives in many combinations Sequencing of the gel PCR products facilitates determination of the breakpoint sequence. Sequencing the MLL translocation. C.Meyer et al. Leukemia 2005

10. MLL Taqman probe R Q Real-time Quantitative PCR MLLForwardprimer MLL gene Partner gene MLL Reverse primer

11. MLL Taqman probe R Q Real-time Quantitative PCR MLL Forward primer MLL gene Partner gene MLL Reverse primer

12. 1/10 1/100 1/1,000 1/10,000 Patient dilution series Real-time Quantitative PCR Typical dilution series – Logarithmic dilutions of diagnostic DNA in normal mononuclear DNA

13. Logarithmic dilutionseries Pre OCTADA(D) sample Normal MNC DNA Real-time Quantitative PCR Example of MRD negative patient

14. Logarithmic dilutionseries Pre OCTADA(D) samples Real-time Quantitative PCR Example of MRD positive patient

15. Clinical outcome • MRD negative at pre-OCTADA(D): continue on the course of chemotherapy • MRD positive at pre-OCTADA(D): put forward for a bone marrow transplant

16. Questions and Acknowledgements • Thanks to • Jerry Hancock, Paul Archer, Paula Waits, Kayleigh Templeman, Helen Williamson & Adiela Chudliegh • Nicola Wolstenholme • Phil Ancliff, Gianni Cazzaniga & Delwar Hussein • Claus Meyer • Any questions? • So long as they are not on LDI-PCR!